Current vaccination technology is based almost exclusively on systemic vaccination techniques wherein the vaccine is injected into the subject to be vaccinated. Only certain live/attenuated vaccines, such as Sabin polio vaccine, may be taken orally.
The advantages of oral immunization techniques are several fold. For instance, it is self-evident that a vaccine which may be fed to subjects is easier to administer on a large scale in the absence of specialized equipment, especially to subjects which may be difficult to handle or even locate, such as livestock and wild animals. The spread of infection by the re-use of needles in developing countries would thereby be avoided. Furthermore, an oral vaccine may be provided in the form of an edible solid, which is easier to handle under extreme conditions and is more stable than the liquid suspensions as currently used.
Moreover, delivery of immunogens to a mucosal membrane, such as by oral or intranasal vaccination, would permit the raising of a secretory immune response.
The secretory immune response, mainly Iga-mediated, appears to be substantially separate from a systemic immune response. Systemic vaccination is ineffective for raising a secretory immune response. This is a considerable disadvantage when considering immunization against pathogens, which often enter the subject across a mucosal surface such as the gut or lung.
Unfortunately, it is not possible to raise a secretory immune response to the vast majority of antigens simply by exposing mucosal surfaces to such antigens. Furthermore, no adjuvant capable of eliciting a secretory immune response to a given antigen is currently available.
The apparent difficulty is largely due to a phenomenon known as oral tolerance. The linings of the gut and the lungs are naturally tolerant to foreign antigens, which prevents an immune response being raised to ingested or inhaled substances, such as food and airborne particulate matter.
The ADP-ribosylating bacterial toxins, namely diphtheria toxin, pertussis toxin (PT), cholera toxin (CT), the E. coli heat-labile toxin (LT1 and LT2), Pseudomonas endotoxin A, C. botulinum C2 and C3 toxins as well as toxins from C. perfringens, C. spiriforma and C. difficile are potent toxins in man. These toxins are composed of a monomeric, enzymatically active A subunit which is responsible for ADP-ribosylation of GTP-binding proteins, and a non-toxic B subunit which binds receptors on the surface of the target cell and delivers the A subunit across the cell membrane. In the case of CT and LT, the A subunit is known to increase intracellular cAMP levels in target cells, while the B subunit is pentameric and binds to GM1 ganglioside receptors.
In 1975 and 1978 observations were made which demonstrated that CT is able to induce mucosal and systemic immunity against itself when administered intraduodenally (i.e. to a mucosal surface). The membrane-binding function of CT was shown to be essential for mucosal immugenicity, but cholera toxoid, also known as the B subunit of CT (CTB) was inactive in isolation (Pierce and Gowans, J. Exp. Med 1975; 142: 1550; Pierce, J. Exp Med 1978; 148: 195-206).
Subsequently, it was demonstrated that CT induces systemic and mucosal immunity to co-administered antigens, in other words functions as a mucosal adjuvant (Elson, Curr. Top. Microbiol. Immunal, 1989; 146: 29; Elson and Ealding, J. Immunol. 1984; 133: 2892-2897; Elson and Ealding, Ibid. 1984; 132: 2736-2741; Elson et al., J. Immunol. Methods 1984; 67: 101-118; Lycke and Homgren, Immunology 1986; 59: 301-338).
The experiments referred to above were conducted in mice, which are comparatively resistant to the toxic effects of CT. In contrast, wild-type CT is extremely toxic to humans, rendering the use of CT having any substantial residual toxicity as a mucosal adjuvant in humans entirely out of the question.
Two approaches have been taken in the prior art to address the problem of CT toxicity. The first approach has involved the use of CTB as a mucosal adjuvant. CTB is entirely non-toxic.
In one series of experiments, CTB was covalently coupled to horseradish peroxidase (HRP) and administered to mice intraduodenally. This gave rise to a powerful mucosal immune response to HRP (McKenzie and Halsey, J. Immunol 1984; 133: 1818-1824).
This result has subsequently been partially confirmed with other antigens (Liang et al., J. Immunol 1988; 141: 1495-1501; Czerkinsky et al., Infect. Immun. 1989; 57: 1072-1077). The same principle has also been established to be effective when chimeric antigens produced by gene fusion to sequences encoding CTB have been tested (Dertzbaugh and Elson, Infect. Immun. 1993; 61: 384-390; Dertzbaugh and Elson, Ibid. 1993; 61: 48-55; Sanchez et al., Res. Microbiol 1990; 141: 971-979; Holmgren et al., Vaccine 1993; 11: 1179-1184).
However, the production of chimeric or coupled antigens introduces a further step in the preparation of suitable vaccines, which essentially requires that antigens be prepared in a form conjugated with CTB especially for oral use. It would be for simpler and more economical if the adjuvant could be administered in simple admixture with the antigen.
An adjuvant effect for co-administered CTB has been alleged in a number of publications (Tamura et al., J. Immunol 1992; 149: 981-988; Hirabayashi et al., Immunology 1992; 75: 493-498; Gizurarson et al., Vaccine 1991; 9: 825-832; Kikuta et al., Vaccine 1970; 8: 595-599; Hirabayashi et al. Ibid. 1990; 8; 243-248; Tamura et al., Ibid. 1989; 7: 314-32-; Tamura et al., Ibid. 1989; 7: 257-262; Tamura et al., Ibid 1988; 6: 409-413; Hirabayashi et al., Immunology 1991; 72: 329-335 Tamura et al., Vaccine 1989; 17: 503-505).
However, a number of aspects of the observations reported above were not entirely convincing. For example, it was noted that the adjuvant effect ascribed to CTB was not H-2 restricted. It is known, however, that immune response to CTB is H-2 restricted (Elson and Ealding, Eur. J. Immunol. 1987; 17: 425-428). Moreover, the alleged adjuvant effect was observed even in individuals already immune to CTB.
Other groups were unable to observe any mucosal adjuvant effect attributable to CTB (Lycke and Holmgren, Immunology 1986; 59: 301-308; Lycke et al., Eur. J. Immunol. 1992; 22: 2277-2281). Experiments with recombinant CTB (Holmgren et al., Vaccine 1993; 11: 1179-1183) confirmed that the alleged effect is largely if not exclusively attributable to low levels of contamination of CTB preparations with CT.
Thus, it is presently accepted that CTB is not useful as a mucosal adjuvant.
A second approach to eliminating the toxicity of CT has been to mutate the CT holotoxin in order to reduce or eliminate its toxicity. The toxicity of CT resides in the A subunit and a number of mutants of CT and its homologue, LT, comprising point mutations in the A subunit are known in the art. See, for example, International Patent Application WO92/19265 (Amgen). It is accepted in the art that CT and LT are generally interchangeable, showing considerable homology.
However, the only mutant so far tested for mucosal adjuvanticity, an LT mutant having a Glu→Lys mutation at position 112, was found to be inactive as a mucosal adjuvant (Lycke et al; Eur. J. Immunol. 1992; 22: 2277-2251; Holmgren et al., Vaccine 1993; 11: 1179-1183). The authors of these publications conclude that there is a link between the ADP ribosylating activity of CT and/or LT and the adjuvant activity. It appears from these publications, therefore, that CTB or a non-toxic mutant of CT or LT would not be active as a mucosal adjuvant.